Restriction endonucleases belong to the class of enzymes called nucleases which degrade or cut single or double stranded DNA. A restriction endonuclease acts by recognizing and binding to particular sequences of nucleotides (the ‘recognition sequence’) along the DNA molecule. Once bound, the endonuclease cleaves the molecule within or to one side of the recognition sequence. The location of cleavage may differ among various restriction endonucleases, though for any given endonuclease the position is fixed. Different restriction endonucleases have different affinity for recognition sequences. More than two hundred restriction endonucleases recognizing unique specificities have been identified among thousands of bacterial and archaeal species that have been examined to date.
Restriction endonucleases are classified on the basis of their composition and cofactor requirements, the nature of target sequence, and the position of the site of DNA cleavage with respect to the target sequence (Yuan, R. Ann. Rev. Biochem., 50:285-315 (1981)). Currently three distinct, well-characterized classes of restriction endonucleases are known (I, II and III). Type I enzymes recognize specific sequences, but cleave randomly with respect to that sequence. The type III restriction endonucleases recognize specific sequences, cleave at a defined position to one side of that sequence, but never give complete digestion. Neither of these two kinds of enzymes is suitable for practical use. The type II restriction endonucleases recognize specific sequences (4-8 nucleotides long) and cleave at a defined position either within or very close to that sequence. Usually they require only Mg2+ ions for their action. When they are purified away from other bacterial components, type II restriction endonucleases can be used in the laboratory to cleave DNA molecules into specific fragments. This property allows the researcher to manipulate the DNA molecule and analyze the resulting constructions.
Bacteria tend to possess at most, only a small number of restriction endonucleases per isolate. The restriction endonucleases are designated by a three-letter acronym derived from the name of organism in which they occur (Smith and Nathans, J. Mol. Biol. 81:419-423 (1973)). The first letter comes from the genus, and the second and third letters come from the species. Thus, a strain of the species Deinococcus radiophilus for example, synthesizes three different type II restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences TTTAAA, PuGGNCCPy and CACNNNGTG, respectively. Escherichia coli RY13, on the other hand, synthesizes only one type II restriction enzyme, EcOR1, which recognizes the sequence GAATTC (Roberts R. J and Macelis D., Nucl. Acids Res., 28:306-7 (2000)).
A second component of bacterial and archaeal restriction systems are the modification methylases (Roberts and Halford, in ‘Nucleases’, 2nd ed.'s, Linn et al., ed.'s, p. 35-88 (1993)). These enzymes are complementary to restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign, invading DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one or other of the nucleotides within the sequence by the addition of a methyl group. Following methylation, the recognition sequence is no longer cleaved by the restriction endonuclease. The DNA of a bacterial cell is modified by virtue of the activity of its modification methylase, and is therefore insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified, and therefore identifiably foreign DNA, that is sensitive to restriction endonuclease recognition and cleavage.
It is thought that in nature, type II restriction endonucleases cleave foreign DNA such as viral and plasmid DNA when this DNA has not been modified by the appropriate modification enzyme (Wilson and Murray, Annu. Rev. Genet. 25:585-627 (1991)). In this way, cells are protected from invasion by foreign DNA. Thus, it has been widely believed that evolution of type II restriction modification systems has been driven by the cell's need to protect itself from infection by foreign DNA (the cellular defense hypothesis).
With the advent of genetic engineering technology, it is now possible to clone genes and to produce the proteins and enzymes that they encode in greater quantities than are obtainable by conventional purification techniques. The key to isolating clones of restriction endonuclease genes is to develop a simple and reliable method to identify such clones within gene libraries. One potential difficulty is that some restriction endonuclease and methylase genes may not express in E. coli due to differences in the transcriptional and translational machinery of the source organism and of E. coli, such as differences in promotor or ribosome binding sites or the codon composition of the gene. The isolation of the methylase gene requires that the methylase express well enough in E. coli to fully protect at least some of the plasmids carrying the gene. The isolation of the endonuclease in active form requires that the methylase express well enough to protect the host DNA fully, or at least enough to prevent lethal damage from cleavage by the endonuclease. Another obstacle to cloning restriction-modification systems lies in the discovery that some strains of E. coli react adversely to cytosine or adenine modification; they possess systems that destroy DNA containing methylated cytosine (Raleigh and Wilson, Proc. Natl. Acad. Sci., USA 83:9070-9074, (1986)), or methylated adenine (Heitman and Model, J. Bact. 196:3243-3250, (1987)); Raleigh, et al., Genetics, 122:279-296, (1989)) Waite-Rees, et al., J. Bacteriology, 173:5207-5219 (1991)). Cytosine-specific or adenine-specific methylase genes cannot be cloned easily into these strains, either on their own, or together with their corresponding endonuclease genes. To avoid this problem it is necessary to use mutant strains of E. coli (McrA−and McrB−or Mrr−) in which these systems are defective.
Several approaches have been used to clone restriction genes into E. coliL 
1) Selection Based on Phage Restriction
The first cloned systems used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Molec. Gen. Genet 178:717-719, (1980)); HhaII: Mann et al., Gene 3:97-112, (1978)); PstI: Walder et al., Proc. Nat Acad. Sci. 78:1503-1507, (1981)). Since the presence of restriction-modification systems in bacteria enable them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from libraries that have been exposed to phage. This method has been found, however, to have only limited value. Specifically, it has been found that cloned restriction-modification genes do not always manifest sufficient phage resistance to confer selective survival under standard conditions.
2) Selection Based on Vector Modification
A second approach which is being used to clone a growing number of systems, involves selection for an active methylase gene (refer to U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acid. Res. 13:6403-6421, (1985)). Since restriction and modification genes are often closely linked, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead may yield only the methyltransferase gene (BspRI: Szomolanyi et al., Gene 10:219-225, (1980); BcnI: Janulaitis et al, Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119, (1983); and MspI: Walder et al., J. Biol. Chem. 258:1235-1241, (1983)).
3) Sub-cloning of Natural Plasmids
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning plasmids (EcoRVI: Bougueleret et al., Nucl. Acid. Res. 12: 3659-3676, (1984); PaeR7I: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406, (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509, (1985)).
4) Multi-step Cloning
Sometimes the straight-forward methylase selection method fails to yield a methylase (and/or endonuclease) clone due to various obstacles. See, e.g., Lunnen, et at., Gene, 74(1):25-32 (1988). One potential obstacle to cloning restriction-modification genes lies in trying to introduce the endonuclease gene into a host not already protected by modification. If the methylase gene and endonuclease gene are introduced together as a single clone, the methylase must protectively modify the host DNA before the endonuclease has the opportunity to cleave it. On occasion, therefore, it might only be possible to clone the genes sequentially, methylase first then endonuclease (see, U.S. Pat. No. 5,320,957).
5) Selection Based on Induction of the DNA-damage-inducible SOS Response
Another method for cloning methylase and endonuclease genes is based on a colorimetric assay for DNA damage (see, U.S. Pat. No. 5,492,823). When screening for a methylase, the plasmid library is transformed into a sensitive host E. coli strain such as AP1-200. The expression of a methylase will induce the SOS response in an E. coli strain which is McrA+, McrBC+, or Mrr+. The AP1-200 strain is temperature sensitive for the Mcr and Mrr systems and includes a lacZ gene fused to the damage inducible dinD locus of E. coli. The detection of recombinant plasmids encoding a methylase or endonuclease gene is based on induction at the restrictive temperature of the lacZ gene. Transformants encoding methylase genes are detected on LB agar plates containing X-gal as blue colonies. (Piekarowicz, et.al., Nucleic Acids Res. 19:1831-1835, (1991) and Piekarowicz, et.al. J. Bacteriology 173:150-155 (1991)). Likewise, the E. coli strain ER1992 contains a dinD1-Lac Z fusion but is lacking the methylation dependent restriction systems McrA, McrBC and Mrr. In this system (called the “endo-blue” method), the endonuclease gene can be detected in the absence of it's cognate methylase when the endonuclease damages the host cell DNA, inducing the SOS response. The SOS-induced cells form deep blue colonies on LB agar plates supplemented with X-gal. (Fomenkov, et.al. Nucleic Acids Res. 22:2399-2403 (1994) and U.S. Pat. No. 5,498,535).
6) N-terminal-sequence-based Degenerate Inverse PCR Method
It may occur that a modification methyltransferase gene cannot be identified (see, U.S. Pat. No. 5,945,288), or that a methylase gene can be identified but the open reading frame specifying the restriction endonuclease is uncertain. In these cases, an additional procedure for identifying the gene for the endonuclease specifically can be applied when the restriction endonuclease can be purified in sufficient quantity and purity from the original organism. In this method, the restriction endonuclease is purified to substantial homogeneity and subjected to polypeptide sequencing. The polypeptide sequence obtained is reverse-translated into DNA sequence and degenerate PCR primers can be designed to amplify a portion of the endonuclease gene from genomic DNA of the original organism or from a gene library made therefrom. The DNA sequence of the complete genes can be obtained by methods dependent on Southern blot analysis or by further direct or inverse PCR methods. If the cognate methyltransferase gene cannot be obtained or cannot be expressed, the stability and utility of the solo restriction endonuclease clone will usually be severely compromised.
It may occur that genes for both the methyltransferase and the restriction endonuclease of a particular system can be obtained by the methods described above, but nevertheless establishment of a usable strain for enzyme production is problematic. Frequently the difficulty is with expression of the methyltransferase gene at a suitable level. This is particularly true with method (6). Such clones sometimes can be stabilized by using heterospecific methyltransferase genes, which were not associated with the endonuclease gene in the original host but which recognize the same or a related sequence and prevent the endonuclease from cleaving its recognition sequence (see, U.S. Pat. No. 6,048,731).
It may occur that there is no suitable heterospecific methyltransferase available, and the degree of protection conferred on the host by the cognate methyltransferase is inadequate; or it may occur that apparently adequate levels of methyltransferase can be obtained but such level is toxic to the cell, resulting in strains that cannot be stored; or it may occur that protection is apparently adequate and the protected strain is viable, but the combination of the methyltransferase and the endonuclease genes gives a strain that does not express detectable endonuclease; or it may occur that protection is apparently adequate, but the combination of the methyltransferase and the endonuclease genes gives a strain that expresses detectable endonuclease, but is not sufficiently stable to make commercially useful levels of enzyme.
Many factors can be imagined that might alter the requisite level of enzyme needed for effective protection of the host cell from cleavage by a restriction endonuclease. Such factors include rapid growth, during which more DNA copies are present in the cell than are present during the stationary phase of growth; recovery from a resting state, during which time new synthesis of the modification methyltransferase may be required before new synthesis of the restriction endonuclease begins; starvation of various sorts, during which time levels of required DNA methyltransferase cofactors such as S-adenosylmethionine may be altered; and special physiological states, such as DNA damage or other physiological insults. In addition, levels of methyltransferase can potentially be too high and become toxic, for example by binding to or methylating extraneous sites related to the cognate site and thus interfering with the reading of the DNA sequence by regulatory or DNA-condensing proteins. Thus, the absolute level of expression of the methyltransferase may need to fluctuate in response to conditions over the life of a culture, in order to be indefinitely perpetuated.
This need for a fine level of control is not unique to modification methyltransferases. Over the course of 50 years of study, many detailed regulatory schemes have been described for various sorts of functions, such as catabolic and anabolic gene sets that break down nutrients (lac, ara, gal) or synthesize essential compounds (trp, his), or response to stressors (the DNA damage response, the heat shock response). These regulatory effects are mediated by changes in promoter activity (by activators or repressors), in transcript stability (by retroregulatory elements), by alteration of translation levels (by attenuation or translational coupling), for example. Despite this high level of understanding, it is not straighforward to anticipate in advance how the demand for a function will change with physiological changes and how to achieve the desired level of a function.
Because purified restriction endonucleases, and to a lesser extent, modification methylases, are useful tools for characterizing genes in the laboratory, there is a commercial incentive to obtain bacterial strains through recombinant DNA techniques that synthesize these enzymes in abundance. Such strains would be useful because they would simplify the task of purification as well as providing the means for production in commercially useful amounts.